Targeted, NIR imaging agents for therapy efficacy monitoring, deep tissue disease demarcation and deep tissue imaging

ABSTRACT

Compounds and methods related to NIR molecular imaging, in-vitro and in-vivo functional imaging, therapy/efficacy monitoring, and cancer and metastatic activity imaging. Compounds and methods demonstrated pertain to the field of peripheral benzodiazepine receptor imaging, metabolic imaging, cellular respiration imaging, cellular proliferation imaging as targeted agents that incorporate signaling agents.

This divisional application claims priority to U.S. patent applicationSer. No. 11/149,602 filed Jun. 10, 2005, now U.S. Pat. No. 7,754,884,which claims priority to U.S. patent application No. 60/641,091 filed onJan. 3, 2005, the contents of which are incorporated herein byreference.

PRIORITY INFORMATION

This application claims priority to U.S. Patent Application No.60/641,091, the contents of which are incorporated herein by reference.

FIELD OF THE INVENTION

This invention relates generally to the field of molecular imaging, andmore specifically to the field of functional imaging, including glucosetransporters, thymidine kinase activity, and peripheral benzodiazepinereceptors as targeted agents that incorporate near-infrared fluorophoresas signaling agents.

BACKGROUND OF THE INVENTION

Current state-of-the-art detection and surgical resection tools used incancer treatment are insufficient. Early stage disease can be missed,resection can be incomplete and these two factors alone are majorcontributors to morbidity and mortality. Outcomes are intrinsicallylinked to disease detection and treatment efficacy. Therefore,improvement in the detection of early cellular changes, as well asenhanced visualization of diseased tissue, is of paramount importance.

Optical methods continue to provide a powerful means for studying celland tissue function. Recent discoveries in Molecular Imaging (MI) arecertain to play a vital role in the early detection, diagnosis, andtreatment of disease. MI will also aid in the study of biological andbiochemical mechanisms, immunology, and neuroscience. MI agents commonlyconsist of a signaling moiety (fluorophore, radioisotope or Gd³⁺ ion)and a targeting functionality such as an antibody or peptide, sugar or aperipheral benzodiazepine receptor (PBR) ligand. NIR molecular imagingagents are particularly attractive due to the inherently low water andtissue absorption in the NIR spectral region. Additionally, the lowscattering cross-section and lack of autofluorescence background in thenear infrared (NIR) region facilitate deep penetration andhigh-resolution images from small interrogated volumes.

While glandular and secretory tissues are normally rich in PBR, otherquiescent tissue ordinarily express PBR at relatively low levels.Primarily spanning the bi-layered mitochondrial membrane, the PBR isexpressed almost ubiquitously and thought to be associated with manybiological functions including the regulation of cellular proliferation,immunomodulation, porphyrin transport, heme biosynthesis, aniontransport, regulation of steroidogenesis and apoptosis. Given theimportance of PBR toward regulating mitochondrial function, it is notsurprising that PBR density changes have been observed in acute andchronic neurodegenerative states in humans, as well as numerous forms ofcancer. For example, temporal cortex obtained from Alzheimer's patientsshowed an increase in PBR, and correlations with Huntington's disease,multiple sclerosis and gliosis have been demonstrated. Breast cancergenerally demonstrates increased PBR expression and represents anotherpotentially attractive target, especially in the NIR. The development ofhigh affinity ligands for PBR (such as, for example, PK-11195, Ro5-4864,DAA1106, and DAA1107) has made non-invasive imaging modalities moresuitable.

Other functional imaging targets include the glucose transporter andthymidine kinase 1. By targeting the glucose transporter,[¹⁸F]-fluoro-deoxyglucose (FDG) has been successfully employed as apositron emission tomography (PET) agent to determine the metabolicstatues (cellular respiration) of suspect tissues. Modestfunctionalization of glucose at the C-2 position does not hinder sugaruptake but does prevent cellular metabolism, therefore glucose agentscan accumulate intracellularly. Since tumor cells metabolize glucose ahigher rate than normal cells, the accumulation of glucose mimics (i.e.FDG and similar agents) can facilitate discrimination of tissues basedon their metabolic status. While FDG imaging certainly has demonstratedutility to the clinical oncologist, the requirement of a cyclotron and aPET scanner somewhat limit its use.

Recently, in effort to improve the specificity of functional imagingagents like FDG, new probes for cellular proliferation imaging have beendeveloped. Targeting the enzyme thymidine kinase 1 (TK1), an enzymeresponsible for DNA replication, [¹⁸F]3′-deoxy-3′ fluorothymidine (FLT)has been shown to be an attractive complement to FDG imaging. Similar toFDG, FLT is not fully metabolized by cells and accumulates in targettissues, making it a promising imaging agent for rapidly proliferatingtissues. When used in combination with FDG, clinical imaging of diseasedtissue has the ability to be highly sensitive and specific.

It has been shown that NIR emitting Ln-Chelates can be prepared openingthe avenue to complexes with spectral properties more compatible withbiological imaging such as visible absorption, NIR emission andmicrosecond-long emission lifetimes. These complexes have high molarabsorptivity and have luminescent lifetimes in the microsecond regimeallowing temporal rejection of noise.

The present inventors have demonstrated the synthesis and utility ofEu-PK11195 and Gd-PK11195. Others have prepared PK11195 as a PET agentfor use in humans. A NIR Pyropheophorbide agent has been reported forimaging glucose transporters, however this agent was notspectroscopically optimized for deep tissue in-vivo imaging (ex. 679 nm,em. 720 nm). At present, the authors are unaware of any NIR imagingagents based on thymidine imaging.

SUMMARY OF THE INVENTION

The peripheral benzodiazepine receptor (PBR) has been shown anattractive target for contrast-enhanced imaging of disease. SeePublication No. 2003/0129579, incorporated herein by reference.Embodiments of the present invention include PBR targeted agents whichincorporate near-infrared (NIR) fluorophores as signaling agents.Aspects of the present invention include a previously unknown class ofNIR absorbing/emitting PBR targeted contrast agents which utilize aconjugable form of PK11195 as a targeting moiety.

Additionally, aspects of the present invention include the synthesis ofNIR-metabolic and proliferation probes. The authors report a sacharideagent suitable for metabolic imaging in similar fashion to ¹⁸FDG and aNIR-thymidine probe suitable for imaging cellular proliferation (DNAsynthesis). The NIR contrast agents disclosed herein are suitable foroptical imaging using spectral and time-gated detection approaches tomaximize the signal-to-background ratio. High molar extinction dyes thatabsorb and emit in the NIR, such as IRdye800CW™ (available from LiCOR)and CY7 (Amersham), as well as NIR Lanthanide chelates are demonstrated.Since thymidine, PK11195 and other PBR ligands have been suggested astherapeutic agents, the molecules demonstrated here could also be usefultherapeutics which also offers direct monitoring of dose delivery andtherapeutic efficacy.

With absorption and emission closer to the tissue transparency window(780 nm, 830 nm respectively), the dyes reported here are much moresuited for in-vivo imaging. Additionally, no one has demonstrated NIRPBR ligands for imaging PBR expression and/or therapy.

Thus, one aspect of the present invention is a method of imaging amolecular event in a sample, the method steps comprising administeringto the sample a probe having an affinity for a target. The probe has atleast one of a ligand/signaling agent combination, or conjugable form ofa ligand/signaling agent combination. After the probe is administered, asignal from the probe may be detected. In embodiments of the presentinvention, the sample can be at least one of cells, tissue, cellulartissue, serum, cell extract, bodily fluids. The bodily fluids may be,for example, breast milk, sputum, vaginal fluids, urine.

Another aspect of the present invention is a method of measuring glucoseuptake. This embodiment comprises the steps of administering to a samplea conjugate, the conjugate comprising a conjugable glucosamine compoundand a signaling agent; and then detecting a signal from said conjugate.In embodiments of the present invention, the sample is at least one ofcells, tissue, cellular tissue, serum, cell extract, bodily fluids.

Another aspect of the present invention is a method of quantifying theprogression of a disease state progression that includes the steps of(a) administering to a first sample a conjugate that comprises aconjugable deoxythymidine compound and a signaling agent; (b) detectinga signal from the conjugate; (c) after a period of time from step (b),administering to a second sample a conjugate, (d) detecting a secondsignal; and (e) comparing the first signal with the second signal todetermine the progress of a disease state. Again examples of the sampleare at least one of cells, tissue, cellular tissue, serum, cell extract,bodily fluids.

Another aspect of the present invention is the above method, where theconjugate includes a peripheral benzodiazepine affinity ligand orconjugable form thereof and a signaling agent.

Another aspect of the present invention is the above method, where theconjugate includes a glucosamine compound and a signaling agent.

In the above embodiments and other embodiments of the present invention,the administration step is in vivo or in vitro.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a color photograph that shows white light and fluorescencepictures of dosed cells and un-dosed cells in accordance with thepresent invention, and is further discussed in Example 6, below. PictureA is a white light picture of dosed cells, Picture B is a fluorescencepicture of dosed cells, Picture C is a white light picture of un-dosedcells, and Picture D is a fluorescence picture of un-dosed cells.

FIG. 2 is a color photograph that shows white light and fluorescencepictures of dosed cells and un-dosed cells in accordance with thepresent invention, and is further discussed in Example 7, below. PictureA is a white light picture of dosed cells, Picture B is a fluorescencepicture of dosed cells, Picture C is a white light picture of un-dosedcells, and Picture D is a fluorescence picture of un-dosed cells.

FIG. 3 is a color photograph that shows in vivo cancer imaging of asmall laboratory animal.

FIG. 4 is a color photograph that shows in vivo neurodegenerativeimaging of a small laboratory animal.

DESCRIPTION OF THE INVENTION

Embodiments of the present invention include NIR agents for the PBRbased on NIR dyes, including Lanthanide chelates. Additionally,complimentary imaging agents are disclosed using a novel NIR sacharideand NIR thymidine agent. Aspects of the present invention include bothbeing used separately, as well as where the agents are used together asa cocktail whereby both PBR expression and metabolic and or cellularproliferation status could be simultaneously monitored in-vivo.

PBR ligands such as PK11195 have been suggested as therapeutic agents.Mitochondria localized anti-death proteins of the Bcl-2 family play acentral role in inhibiting apoptosis and therefore present therapeutictargets. PBR shares a close physical association with the permeabilitytransition pore complex (PTPC) and binding of PK11195 has been shown tocause Bcl-2 resistant generation of oxidative stress. The agentsreported here are unique in that they facilitate in-vivo monitoring oftherapeutic delivery and efficacy.

As stated above, aspects of the present invention include methods ofimaging a molecular event. in a sample, the method steps comprisingadministering to the sample a probe having an affinity for a target. Theprobe has at least one of a ligand/signaling agent combination, orconjugable form of a ligand/signaling agent combination. One suchligand/signaling agent combination comprises PBR ligands, or conjugableforms thereof.

Examples of the PBR ligands of the present invention include conjugableforms, or conjugable analogs of the following compounds:

For the purposes of the present invention, the term analog encompasesisomers, homologs, or other compounds sufficiently resembling the basecompound in terms of structure and do not destroy activity. “Conjugableforms,” “conjugable compounds,” and similar terms describe a form of thecompound that can readily form a covalent form a covalent bond with asignaling agent such as an IR dye.

For exemplary purposes, conjugable forms of PK11195, above, include atleast the following compounds, and/or analogs or derivatives thereof:

wherein R is H or alkyl, n is 0-10, and “halo” is fluorine, chlorine,bromine, iodine. In other embodiments of the present invention, halo ischlorine.

The term “halo” or “halogen,” as used herein, includes radio isotopes ofhalogen compounds, such as I¹²¹ and F¹⁹.

Additionally, for exemplary purposes, conjugable forms of Ro5-4864include the following and/or analogs or derivatives thereof:

wherein the variables are defined above. In other embodiments of thepresent invention, halo is chlorine.

Additionally, for exemplary purposes, conjugable forms of DAA1106include the following and/or analogs or derivatives thereof:

wherein the variables are defined above, and:

In other embodiments of the present invention, halo is chlorine orfluorine.

Additionally, for exemplary purposes, conjugable forms of SSR180575include the following and/or analogs or derivatives thereof:

wherein the variables are defined above. In other embodiments of thepresent invention, halo is chlorine.

Non-limited examples of PBR ligands and signaling moieties include thefollowing compounds:

or an analog thereof, wherein R₁ is a signaling moiety; “halo” isfluorine, chlorine, bromine, iodine; and n is 0-10.

Preferably, in the above examples, the signaling moiety is a dye.

Additionally, other aspects of the present invention includeNIR-sacharide agents suitable for metabolic imaging in similar fashionto ¹⁸FDG. Given the ubiquitous clinical use of ¹⁸FDG, 2-deoxyglucosederivatives have been extensively biologically characterized. SeeCzernin, J.; Phelps, M. E. Annu Rev Med 2002, 53, 89-112. Thesederivatives are useful metabolic imaging agents given the overexpressionof glucose transporters and increased hexokinase activity in tumors. SeeMedina, R. A.; Owen, G. I. Biol Res 2002, 35, 9-26. 2-deoxyglucoseimaging agents are incorporated into cells via the glucose transporterand are subsequently phosphorylated by hexokinase. In phosphorylatingthe probe, the neutral molecule becomes anionic and membraneimpermeable. Functionalization at the 2-position prevents furthermetabolism, and thus the probe is trapped in the cells, with furtheruptake leading to significant accumulation. See Zhang, M.; Zhang, Z. H.;Blessington, D.; Li, H.; Busch, T. M.; Madrak, V.; Miles, J.; Chance,B.; Glickson, J. D.; Zheng, G. Bioconjugate Chem 2003, 14, 709-714.

Additionally, other aspects of the present invention include aNIR-thymidine probe for monitoring cellular proliferation, similar infashion to [¹⁸F]3′-deoxy-3′ fluorothymidine (FLT). FLT has been usedclinically and extensively compared to FDG. See Halter et al. GeneralThoracic Surgery 2004, 127, 1093-1099 and Francis et al. Eur J Nucl MedMol Imaging 2003, 30, 988-994. In proliferating cells, FLT metabolismtakes place within the anabolic arm of the DNA salvage pathway. TK1controls entry into the salvage pathway and converts FLT to themono-phosphate species. The agent is further phosphorylated, but can notbe incorporated into DNA due to its lack of a hydroxyl group at 3′.

With respect to the signaling agents used in connection with the presentinvention, embodiments include near infrared signaling agents. Alsoincludes are dyes, such as, for example, near-infraredfluorophores/fluorescent dyes. Examples include cyanine dyes which havebeen used to label various biomolecules. See U.S. Pat. No. 5,268,486,which discloses fluorescent arylsulfonated cyanine dyes having largeextinction coefficients and quantum yields for the purpose of detectionand quantification of labeled components.

Additional examples include compounds of the following formulas:

and analogs thereof.

Additional examples include dyes available from Li-Cor, such as IR Dye800CW™, available from Li-Cor.

Additional examples include dyes disclosed in U.S. Pat. No. 6,027,709.

US '709 discloses dyes which have the following general formula:

wherein R is —OH, —CO₂H, —NH₂, or —NCS and each of x and y,independently, is an integer selected from 1 to about 10. In preferredembodiments, each of x and y, independently, is an integer between about2 and 6.

In one embodiment, the dye isN-(6-hydroxyhexyl)N′-(4-sulfonatobutyl)-3,3,3′,3′-tetramethylbenz(e)indodicarbocyanine,which has the formula:

In a second embodiment, the dye isN-(5-carboxypentyl)N′-(4-sulfonatobutyl)3,3,3′,3′-tetramethylbenz(e)indodicarbocyanine, which has the formula:

These two dyes are embodiments because they have commercially availableprecursors for the linking groups: 6-bromohexanol, 6-bromohexanoic acidand 1,4-butane sultone (all available from Aldrich Chemical Co.,Milwaukee, Wis.). The linking groups provide adequate distance betweenthe dye and the biomolecule for efficient attachment without impartingexcessive hydrophobicity. The resulting labeled biomolecules retaintheir solubility in water and are well-accepted by enzymes.

These dyes, wherein R is —CO₂H or —OH can be synthesized, as set forthin detail in the US '709 patent, by reacting the appropriateN-(carboxyalkyl)- orN-(hydroxyalkyl)-1,1,2-trimethyl-1H-benz(e)indolinium halide, preferablybromide, with sulfonatobutyl-1,1,2-trimethyl-1H-benz(e)indole at arelative molar ratio of about 0.9:1 to about 1:0.9, preferably 1:1 in anorganic solvent, such as pyridine, and heated to reflux, followed by theaddition of 1,3,3-trimethoxypropene in a relative molar ratio of about1:1 to about 3:1 to the reaction product and continued reflux. Themixture subsequently is cooled and poured into an organic solvent suchas ether. The resulting solid or semi-solid can be purified bychromatography on a silica gel column using a series ofmethanol/chloroform solvents.

As an alternative, two-step, synthesis procedure, also detailed in U.S.'709, N-4-sulfonatobutyl-1,1,2-trimethyl-1H-benz(e)indole andmalonaldehyde bis(phenyl)mine)-monohydrochloride in a 1:1 molar ratiocan be dissolved in acetic anhydride and the mixture heated. The aceticanhydride is removed under high vacuum and the residue washed with anorganic solvent such as ether. The residual solid obtained is dried andsubsequently mixed with the appropriate N-(carboxyalkyl)- orN-(hydroxyalkyl)-1,1,2-trimethyl-1H-benz(e)indolinium halide in thepresence of an organic solvent, such as pyridine. The reaction mixtureis heated, then the solvent is removed under vacuum, leaving the crudedesired dye compound. The procedure was adapted from the two stepprocedure set forth in Ernst, L. A., et al., Cytometry 10:3-10 (1989).

The dyes also can be prepared with an amine or isothiocyanateterminating group. For example,N-(omega.-amino-alkyl)-1,1,2-trimethyl-1H-benz(e)indolenium bromidehydrobromide (synthesized as in N. Narayanan and G. Patonay, J. Org.Chem. 60:2391-5 (1995)) can be reacted to form dyes of formula 1 whereinR is —NH₂. Salts of these amino dyes can be converted to thecorresponding isothiocyanates by treatment at room temperature withthiophosgene in an organic solvent such as chloroform and aqueous sodiumcarbonate.

These dyes have a maximum light absorption which occurs near 680 nm.They thus can be excited efficiently by commercially available laserdiodes that are compact, reliable and inexpensive and emit light at thiswavelength. Suitable commercially available lasers include, for example,Toshiba TOLD9225, TOLD9140 and TOLD9150, Phillips CQL806D, Blue SkyResearch PS 015-00 and NEC NDL 3230SU. This near infrared/far redwavelength also is advantageous in that the background fluorescence inthis region normally is low in biological systems and high sensitivitycan be achieved.

The hydroxyl, carboxyl and isothiocyanate groups of the dyes providelinking groups for attachment to a wide variety of biologicallyimportant molecules, including proteins, peptides, enzyme substrates,hormones, antibodies, antigens, haptens, avidin, streptavidin,carbohydrates, oligosaccharides, polysaccharides, nucleic acids, deoxynucleic acids, fragments of DNA or RNA, cells and synthetic combinationsof biological fragments such as peptide nucleic acids (PNAs).

In another embodiment of the present invention, the ligands of thepresent invention may be conjugated to a lissamine dye, such aslissamine rhodamine B sulfonyl chloride. For example, a conjugable formof DAA1106 may be conjugated with lissamine rhodamine B sulfonylchloride to form a compound of the present invention.

Lissamine dyes are typically inexpensive dyes with attractive spectralproperties. For example, examples have a molar extinction coefficient of88,000 cm⁻¹ M⁻¹ and good quantum efficient of about 95%. It absorbs atabout 568 nm and emits at about 583 nm (in methanol) with a decentstokes shift and thus bright fluorescence.

Coupling procedures for the PBR ligands and Glucosamine proceed viastandard methods and will be recognized by those skilled in the art. Ingeneral, the nucleophilic N terminus of the targeting moieties arereactive towards activated carbonyls, for example an NHS(N-hydroxysuccinimide ester), sulfonyl chlorides, or other electrophilebearing species. Solvent of choice for coupling reactions can be dyespecific, but include dimethyl sulfoxide (DMSO), chloroform, and/orphosphate buffered saline (PBS buffer). The resulting conjugates,amides, sulfonamides, etc. resist hydrolysis under physiologicalconditions, and are thus useful for in-vivo and in-vitro application.

The following are examples of compounds of the present invention:

The following compound is an example of one of the coupled compoundsdescribed above:

and analogs thereof, wherein n and x are integers from 1 to 10.

As stated above, the compounds of the present invention can be employedas signaling agents in NIR imaging. The resulting signal may be used toimage a molecular event. Non-limiting examples of specific molecularevents associated with the present invention include at least one ofperipheral benzodiazepine expression, cell proliferation, glucoseuptake, epidermal growth factor receptor expression, coronary disease.

Thus, the resulting signal may be used to diagnose a disease state suchas, for example, cancer, neurodegenerative disease, multiple sclerosis,epilepsy, coronary disease, etc. Specifically, brain cancer and breastcancer are two cancers that may be diagnosed with the compounds andmethods of the present invention. Two additional examples arenon-Hodgkin's lymphoma and colon cancer.

Another embodiment of the present invention is a method of measuringglucose uptake. This method comprises, comprises administering to asample a conjugate, the conjugate comprising a conjugable glucosaminecompound and a signaling agent; and detecting a signal from saidconjugate. As in the other methods, the sample is at least one of cells,tissue, cellular tissue, serum or cell extract. An example of aconjugable glucosamine includes the following compound and conjugableanalog thereof:

The administration step may be in vivo administration or in vitroadministration. The in vivo administration step further comprises atleast one time course imaging determination, and in other embodiments,the in vivo administration step further comprises at least one biodistribution determination.

Other embodiments of the present invention include conjugable compoundsassociated with this glucosamine method, specifically including thefollowing:

where R₁ is a signaling moiety, and

and analogs thereof.

EXAMPLES

The following examples are presented purely for exemplary purposes, andas such the material in this section should be considered as embodimentsof the present invention and not to be limiting thereof.

Example 1

This example demonstrates the conjugation of a NIR dye of the presentinvention and a conjugable analog or conjugable form of PK11195 for deeptissue imaging. In this example, IRDye800CW (LiCOR) is coupled toconjugable PK11195.

Dye800CW-PK11195 (Scheme 1)—To a 10 mL round bottom flask, about 196.5μL of a 1 mg/ml conjugable PK11195 solution (DMSO) is mixed with about300 μL of an about 1 mg/mL Dye800CW (DMSO). The reaction proceeds undernitrogen flow for about 1 hour at RT. Reaction progress is monitored viaHPLC and ESI MS.

Yield is about 99% and requires no further purification.

Example 2

This example demonstrates an example of the formulation of aNIR-glucosamine conjugate of the present invention.

Dye800CW-glucosamine (Scheme 2)—To a 10 mL round bottom flask, about 9.3mg sodium methoxide and about 37 mg D-glucosamine hydrochloride arereacted in about 2 mL DMSO. The solution is stirred under nitrogen forabout 3 hours at RT. Next, about 34, of the resulting solution are mixedwith about 150 μL of an about 1 mg/mL Dye800CW/DMSO solution in aseparate 10 mL flask. The mixture is stirred under nitrogen for another1.5 hours at RT.

Reaction progress was monitored via HPLC and ESI MS and the reactionyielded 98% pure conjugate.

Example 3

This example demonstrates the use of compounds of the present inventionin ESI (Electrospray Ionization) mass spectra.

Initially, about 20 μL of the reaction solution of Example 1 is dilutedto about 180 μL using 5 mM ammonium acetate aqueous solution containingabout 0.05% acetic acid. The sample is injected the sample immediatelyinto a Mariner ESI mass spectrometer. Some major instrument settingsare: spray tip at about 3.4 kv, nozzle potential at about 200v,quadrupole temperature at about 150° C. and nozzle temperature at about150° C. Spectra is collected every 100 seconds. In spectrum forDye800W-glucosamine complex, the expected molecular peak is observed at1164Da. In the spectrum for Dye800CW-PK11195 complex, the expectedmolecular peak is observed at 1365.9Da.

Example 4

This example shows a synthetic pathway yielding a conjugable Ro5-4864 ofthe present invention, and conjugation to an imaging agent, such asLanthanide chelate or NIR-dye.

A conjugable form of compounds similar to Ro5-4864 has been previouslyreported (see U.S. Pat. No. 5,901,381) and a synthetic procedure inScheme 3 will be used to synthesize a conjugable form of Ro5-4864. Asolution of KOH in methanol will be treated with a solution of4-chlorophenyl-acetonitrile 1 and 4-chloronitro-benzene 2 in benzene.The mixture will be stirred for 3 hours and then poured to ammoniumchloride solution. Compound 3 will then precipitate out. Compound 4 willbe produced by stirring compound 3 and dimethyl sulfate for 5 hours,followed by being treated with ethanol, water, iron fillings andhydrochloric acid. See Vejdelek Z, Polivka Z, Protiva M. Synthesis of7-Chloro-5-(4-Chlorophenyl)-1-Methyl-1,3-Dihydro-1,4-Benzodiazepin-2-One.Collection of Czechoslovak Chemical Communications 1985; 50:1064-1069.Compound 4 and semicarbazide, after heated to 210° C., will producecompound 5. Compound 7 will be used as a linker to combine compound 5and lanthanide chelate/dye800cw. Compound 7 can be synthesized by thereaction between compound 6 and thionyl chloride. Lanthanide chelate(with carboxylic acid group) or dye800cw (a N-hydroxysuccinimide ester)can then react with compound 7 in basic solution to produce a compoundin the form of compound 8. The chlorine on the signaling part will reactwith N—H group in compound 5 to produce the final imaging agent(compound 9). The product can be further chelated by adding lanthanidechloride solution (LnCl₃, EuCl₃ etc) into product solution with pH 6.5.The synthetic pathway for lanthanide chelate has been reported. SeeGriffin J M M, Skwierawska A M, Manning H C, Marx J N, Bornhop D J.Simple, high yielding synthesis of trifunctional fluorescent lanthanidechelates. Tetrahedron Letters 2001; 42:3823-3825.

Example 5

This Example shows a scheme for the synthesis of a conjugable form ofDAA1106 of the present invention, which cnathen be conjugated to animaging agent.

The synthetic pathway for conjugable DAM106 is shown in Scheme 4.Compound 2 will be obtained by reaction of compound 1 with phenol inDMF. Compound 2 will then be reduced by PtO2 under hydrogen flow inmethanol. Compound 3 can react with acetyl chloride in pyridine toproduce compound 4 after the reaction refluxes for 2 hours. The hydroxylgroup in compound 5 will be substituted by bromide to produce compound6. One hydroxyl group in compound 6 will be deprotected in DMF by Sodiumethanethiolate to produce compound 7. Compound 4 and 7 will then reactin DMF with the presence of sodium hydride. After compound 8 isobtained, the hydroxyl group will be brominated to form compound 9.Conjugable DAA1106 (compound 10) is prepared by treatment of compound 9with hexane-1,6-diamine. The conjugation position on DAM106 isdetermined according to another conjugation that has been done onDAA1106 which did not affect the biological activity of DAA1106. SeeZhang M R, Maeda J, Furutsuka K, Yoshida Y, Ogawa M, Suhara T, et al.[F-18]FMDAA1106 and [F-18]FEDAA1106: Two positron-emitter labeledligands for peripheral benzodiazepine receptor (PBR). Bioorganic &Medicinal Chemistry Letters 2003; 13:201-204. The product should beconjugable to lanthanide chelator in water/DMF/dioxane/TEA mixture. Theconjugate will be further chelated by adding Lanthanide chloridesolution (LnCl₃, EuCl₃ etc) into pH 6.5 product solution.

Example 6

This example shows an example of the synthesis, characterization, andpreliminary cell study for an embodiment of the present invention, adye800cw-DAA1106 conjugation, as well as the conjugation of the PBRligand DAA1106 to a NIR dye, followed by cell uptake.

In this example, dye800CW (5 mg, 4.3 pimp and conjugable DAA1106 (5 mg,10 μmol) is mixed in DMSO (1 mL) in a 10 mL round bottom flask. Thesolution is stirred under argon flow for 10 hours. The reaction schemeis shown in Scheme 5, below. Product is purified through neutral aluminacolumn using 0.1 M triethyl ammonium acetate in 80/20 acetonitrile/watersolution.

Upon preparing dye800CW-DAA1106, absorption and emission spectra(Table 1) are obtained at room temperature with a Shimadzu 1700 UV-visspectrophotometer and ISS PCI spectrofluorometer respectively. The samesample (2 μM) is used for taking both UV and fluorescence spectrum. UVspectrum was scanned from 190 nm to 900 nm with sampling rate of 1 nm.Cuvette path length was 1 cm. Fluorescence sample was excited at 797 nm.Spectrum was collected from 700 nm to 900 nm with scan rate 1 nm/second.Slit width was set to 1.5. Photo multiplier tube (PMT) voltage was at 75watts. Dye800CW-DAA1106 has maximum absorption at 779 nm andfluorescence at 801 nm in methanol.

TABLE 1 Absorption and fluorescence of IRDye800cw-DAA1106 Absorption andfluorescence of dye800CW-DAA1106. Absorption λ_(max) = 779 nm andfluorescence λ_(max) = 801 nm

Regarding cell uptake, C6 glioma cell lines are a widely used cell linein neurobiological research that has high PBR expression. C6 cells wereincubated with 10 μM dye800CW-DAA1106 in culture media for half hour andthen rinsed and re-incubated with saline before imaging. FIG. 1 showswhite light and fluorescence pictures of dosed and un-dosed cells.Instrument used is Nikon epifluorescence microscope equipped with LudlQimaging camera, Nikon S fluor 20x/0.75 objective, mercury lamp and ICGfilter set. Picture B shows cell take-up of dye800CW-DAA1106, whileun-dosed cell (picture D) does not show any significant fluorescence.

Example 7

This example shows the synthesis, characterization and preliminary cellstudy of a lissamine-DAA1106 conjugation. An example of a lissamine dyehas a molar extinction coefficient of 88,000 cm⁻¹ M⁻¹ and good quantumefficient of about 95%. It absorbs at 568 nm and emits at 583 nm (inmethanol) with a decent stokes shift and thus bright fluorescence.

Lissamine rhodamine B sulfonyl chloride (4 mg, 6.9 μmol), conjugableDAM106 (5 mg, 10 pimp and tri-ethylamine (10 μL) was mixed indichloromethane (0.8 mL) in a 10 mL round bottom flask. The solution wasstirred under argon flow for 3 hours. The reaction scheme is shown inScheme 6. Product was purified through column chromatography (silicagel) using 19/1 dichloromethane/methanol solution.

Upon preparing lissamine-DAA1106, absorption and emission spectra (Table2) was obtained with a Shimadzu 1700 UV-vis spectrophotometer and ISSPTI spectrofluorometer at room temperature. The same sample (2 μM) wasused for taking both UV and fluorescence spectrum. UV spectrum wasscanned from 190 nm to 900 nm with sampling rate of 1 nm. Cuvette pathlength was 1 cm. Fluorescence sample was excited at 561 nm. Spectrum wascollected from 700 nm to 900 nm with scan rate 1 nm/second. Slit widthwas set to 1.5. Photo multiplier tube (PMT) voltage was at 75 watts.Lissamine-DAA1106 has maximum absorption at 561 nm and fluorescence at579 nm in methanol.

TABLE 2 Lissamine-DAA1106 absorption and emissition Lissamine-DAA1106absorption and fluorescence Absorption λ_(max) = 561 nm and fluorescenceλ_(max) = 579 nm

C6 cells were incubated with 10 μM lissamine-DAA1106 in culture mediafor half hour and then rinsed and re-incubated with saline beforeimaging. FIG. 2 shows white light and fluorescence pictures of dosed andun-dosed cells. Instrument used was Nikon epifluorescence microscopeequipped with Ludl Qimaging camera, Nikon S fluor 20x/0.75 objective,mercury lamp and Texas red filter set. Picture B shows cell take-up oflissamine-DAA1106 at perinuclear location. This observation was expectedsince PBR is a mitochondrial protein. Un-dosed cells (picture D)exhibited no fluorescence.

Example 8

This example shows an example of a synthetic pathway yielding aconjugable form of a SSR180575 compound of the present invention.

Starting from m-chloroaniline, which was diazotised and coupled withethyl α—methylacetoacetate, the azo-ester was converted into ethylpyruvate m—chlorophenylhydrazone 1 (the Japp-Klingeman reaction).Polyphosphoric acid facilitated the conversion to molecule 2. Next,N-methylation with dimethylcarbonate in presence K₂CO₃ yielded the ester3 and was treated with hydrazine and converted into hydrazide 4. Thering was closed in the presence of POCl₃ and compound 5 was obtained.N-phenylation with using PhI and CuI (as catalyst) provide compound 6.Mild hydrolysis with dilute KOH in EtOH yield acid 7. To conjugatedmono-N-BOC protected N-methyl-1,6-hexanediamine to 7 used BOP. Removalof protecting group with TFA in CH₂Cl₂ is yields 8.

Example 9

Example 9 demonstrates specific, in vivo tumor labeling using a methodof the present invention. A NIR-PK 11195 deep tissue imaging agent wasmade as shown in Example 1. Tumor bearing Smad3 gene knockout mice andcontrol animals were injected with 10 nmoles of the imaging agent andimaged about 14 hours following injection. Specific labeling wasobserved in the abdominal region of tumor animals and clearance in thecontrol animals. This selective uptake is shown in FIG. 3. Apost-imaging autopsy confirmed localization of the imaging agent in theSMAD3 animal.

Example 10

This Example shows NIR-PK 11195 imaging in connection withneurodegenerative processes in experimental autoimmune encephalomyelitis(EAE), the animal model of multiple sclerosis. Additionally, thisExample shows the use of the present invention to monitor theprogression of a disease state. A conjugated imaging agent NIR-PK 11195was made in accordance with Example 1. An EAE induced and controlanimals were injected with NIR-PK 11195 and imaged. EAE animalsdemonstrate strong fluorescence along the spinal column indicatingactivated T cell and macrophage response which signal the onset of thedemylenation processes characteristic to EAE. The EAE/treated mouse wastreated with a curcumin composition.

FIG. 3 shows images associated with this example that confirminsignificant uptake of the imaging agent in the control, but indicatefull onset of a disease state in the EAE mice. Subsequent imaging showsthe progression of the disease after a disease state treatment isadministered.

REFERENCES

Throughout this application, various publications are referenced. Allsuch publications, specifically including the publications listed below,are incorporated herein by reference in their entirety.

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It will be apparent to those skilled in the art that variousmodifications and variations can be made in the present inventionwithout departing from the scope or spirit of the invention. Otherembodiments of the invention will be apparent to those skilled in theart from consideration of the specification and practice of theinvention disclosed herein. It is intended that the Specification andExample be considered as exemplary only, and not intended to limit thescope and spirit of the invention.

Unless otherwise indicated, all numbers expressing quantities ofingredients, properties such as reaction conditions, and so forth usedin the Specification and Claims are to be understood as being modifiedin all instances by the term “about.” Accordingly, unless indicated tothe contrary, the numerical parameters set forth in the Specificationand Claims are approximations that may vary depending upon the desiredproperties sought to be determined by the present invention.

Notwithstanding that the numerical ranges and parameters setting forththe broad scope of the invention are approximations, the numericalvalues set forth in the experimental or example sections are reported asprecisely as possible. Any numerical value, however, inherently containcertain errors necessarily resulting from the standard deviation foundin their respective testing measurements.

1. A compound of the following formula:

or an analog thereof, wherein R₁ is a signaling moiety, “halo” isfluorine, chlorine, bromine, iodine; and n is 0-10.
 2. The compound ofclaim 1, wherein the signaling moiety is a dye.
 3. A compound of claim2, of the following formula:


4. A method of imaging a molecular event in a sample, comprising: (a)administering to said sample a probe having an affinity for a target,the probe comprising at least one of a ligand/signaling agentcombination, or conjugable form of said ligand/signaling agentcombination; and (b) detecting a signal from said probe; wherein saidligand is a compound of the following formula:

or a conjugable analog thereof, wherein R₁ is a signaling agent, “halo”is fluorine, chlorine, bromine, iodine, and n is 0-10.
 5. The method ofclaim 4, wherein said sample is at least one of cells, tissue, cellulartissue, serum, cell extract, bodily fluids.
 6. The method of claim 5,wherein the bodily fluids are breast milk, sputum, vaginal fluids,urine.
 7. The method of claim 4, wherein the administering step is invivo or in vitro.
 8. The method of claim 4, wherein said molecular eventis at least one of peripheral benzodiazepine receptor expression, cellproliferation, glucose uptake, epidermal growth factor receptorexpression, microglial activation, apoptosis, matrix metalloproteinaseactivation.
 9. The method of claim 4, wherein said signaling agent is anear infrared signaling agent.
 10. The method of claim 4, wherein saiddetecting step comprises near infrared detection.
 11. The method ofclaim 1, wherein the near infrared detection includes near infraredimaging.
 12. The method of claim 4, further comprising the step of:analyzing said signal to diagnose a disease state.
 13. The method ofclaim 10, wherein the disease state is cancer, neurodegenerativedisease, cancer, multiple sclerosis, epilepsy.
 14. The method of claim12, wherein the disease state is brain cancer or breast cancer.
 15. Themethod of claim 4, wherein the signaling agent is a cyanine dye.